Over the last two cycles of this RO1 award, our research groups have studied the role of cold storage and post transfusion hemolysis as one of the pathways that define the red blood cell (RBC) storage lesion. As summarized in our progress report, these studies have catalyzed a field of research, encompassing the biophysics that govern hemolysis and microparticle related impairments in NO/ROS equilibrium, studies of the transfusion-recipient physiological and pathological responses, and interactions with unit age in storage. We now focus on a simple observation, that the variability in donor RBC stability and function during cold storage and post-transfusion recovery is more dependent on the donor, than to the actual time the unit is in storage. For example, mouse and human female blood is more stable during storage than male blood, and even more striking, common mutations like sickle cell trait (HbAS) exhibit enhanced storage hemolysis and lower post-transfusion recovery. These observations have informed our overarching hypothesis that evolved variability in genes encoding RBC proteins significantly modulate RBC function and recovery after transfusion. The genetic variability that characterizes RBC protein expression levels and function has evolved in large part as a consequence of endemic malaria, which has exerted significant evolutionary pressure on the human genome. Few studies have explored how these common and rare variants might modulate donor RBCs during storage and post-transfusion, nor how these variants might affect susceptibility to hemolysis in disease. To address this question, in collaboration with the REDS III program, we performed the largest genome-wide association study (GWAS) study to date of over 13,000 healthy human blood donors, comparing high density single nucleotide polymorphisms (SNPs) with RBC hemolysis responses to canonical in vitro osmotic, oxidative and storage stress. These RBC-Omics studies have identified almost 19 candidate SNPs at genome wide significance (P values<10-8) that are associated with enhanced or reduced responses to hemolytic perturbations. In our RO1 renewal we propose to explore the function of these common and rare variant SNPs in vitro in human red cells from recalled donors with identified SNPs (Aim 1), in mouse models of transfusion (Aim 2), and in humans with autologous biotin labeled transfusion-recovery studies (Aim 3). Using validated transfusion models that we have developed over the last 3 years for dye-labeled mouse and human donor RBC transfusions into mice, we will explore post-transfusion recoveries of RBC with conserved (similar sequences in mouse and humans) non-synonymous SNPs implicated to RBC stress hemolysis responses, including SNPs in ANK1, SPTA, G6PD, ESYT2, ITFG3, and PIEZ01. To assess in vivo mechanisms, we will generate transgenic mice with human GWAS SNPs using CRISPR-Cas9. We anticipate that these findings will inform a Precision Transfusion Medicine field, in which RBC donor genotype determines the time limits of blood storage and recovery of cells after storage (i.e. the identification of super donors versus fragile donors).